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  • Q5® High-Fidelity DNA Polymerase

    Description

      
    Q5® High-Fidelity DNA Polymerase is a high-fidelity, thermostable DNA polymerase with 3´→ 5´ exonuclease activity, fused to a processivity-enhancing Sso7d domain to support robust DNA amplification. With an error rate > 100-fold lower than that of Taq DNA Polymerase and 12-fold lower than that of Pyrococcus furiosus (Pfu) DNA Polymerase, Q5 High-Fidelity DNA Polymerase is ideal for cloning and can be used for long or difficult amplicons. Q5 High-Fidelity DNA Polymerase is supplied with an optimized buffer system that allows robust amplification regardless of GC content. The 5X Q5 Reaction Buffer contains 2 mM MgCl2 at final (1X) reaction concentrations and is recommended for most routine applications. For GC-rich targets (≥ 65% GC), amplification can be improved by the addition of the 5X Q5 High GC Enhancer. Q5 High-Fidelity DNA Polymerase is unlike typical, lower fidelity PCR enzymes. To determine the optimal annealing temperatures for a given set of primers, use of the NEB Tm Calculator is highly recommended.

    M0491
    Amplification of a variety of human genomic amplicons from low to high GC content using Q5 High-Fidelity DNA Polymerase. All reactions were conducted using 30 cycles of amplification and visualized by microfluidic LabChip® analysis.

    Product Source

    An E. coli strain that carries the Q5 High-Fidelity DNA Polymerase gene.

    Reagents Supplied

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    Q5® High GC Enhancer5X
    Q5® Reaction Buffer-205X

    Advantages and Features

    Applications

    • High-fidelity PCR
    • Cloning
    • Long or Difficult Amplification
    • High-throughput PCR

    Properties and Usage

    Unit Definition

    One unit is defined as the amount of enzyme that will incorporate 10 nmol of dNTP into acid insoluble material in 30 minutes at 74°C.

    Storage Temperature

    -20°C

    Heat Inactivation

    No

    Unit Assay Conditions

    25 mM TAPS-HCl (pH 9.3 @ 25°C), 50 mM KCl, 2 mM MgCl2, 1 mM β-mercaptoethanol, 200 μM dNTPs including [3H]-dTTP and 400 μg/ml activated Calf Thymus DNA.

    Quality Control

    Quality Control Assays

    The following Quality Control Tests are performed on each new lot and meet the specifications designated for the product. Individual lot data can be found on the Product Summary Sheet/Datacard or Manual which can be found in the Supporting Documents section of this page.
    • Endonuclease Activity (Nicking):
      The product is tested in a reaction containing a supercoiled DNA substrate. After incubation for 4 hours the percent converted to the nicked form is determined by agarose gel electrophoresis.
    • PCR Amplification (DNA Polymerase):

      The polymerase master mix is tested in a polymerase chain reaction (PCR) using a control template and specific primers, resulting in the expected product.

    • PCR Amplification (Enhancer Dependent, GC-rich):

      The polymerase is tested in a polymerase chain reaction (PCR) using a GC-rich control template and specific primers, resulting in the enhancer-dependent production of the expected product.

    • Protein Purity (SDS-PAGE):
      The physical purity is assessed by comparing contaminating protein bands in a concentrated sample to the protein of interest band in a sample of known dilution. The purity is determined by SDS-PAGE.

    References

    1. Anastassia Voronova Erin Coyne, Ashraf Al Madhoun, Joel V. Fair, Neven Bosiljcic, Catherine St-Louis, Grace Li, Sherry Thurig, Valerie A. Wallace, Nadine Wiper-Bergeron, and Ilona S. Skerjanc. (2013). Hedgehog Signaling Regulates MyoD Expression and Activity. J Biol Chem. 288(6), 4389–4404. PubMedID: PMC3567689
    2. Lieve Naesens, Luke Guddat, Dianne Keough, André B.P. van Kuilenburg, Judith Meijer, Johan Vande Voorde and Jan Balzarini (2013). ROLE OF HUMAN HYPOXANTHINE GUANINE 
      PHOSPHORIBOSYLTRANSFERASE IN ACTIVATION 
      OF THE ANTIVIRAL AGENT T-705 (FAVIPIRAVIR). Molecular Pharmacology Fast Forward. 87247
    3. Hicham Bouabe (2013). A Protocol for Construction of Gene Targeting Vectors and Generation of Homologous Recombinant Embryonic Stem Cells. Methods in Molecular Biology . 1064, 337-354.

    Supporting Documents

    Material Safety Datasheets

    The following is a list of Material Safety Data Sheets (MSDS) that apply to this product to help you use it safely. The following file naming structure is used to name these document files: [Product Name] MSDS. For international versions please contact us at info@neb.com.

    Datacards

    The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at info@neb.com or fill out the Technical Support Form for appropriate document.
    1. What are the advantages to using Q5® High-Fidelity DNA Polymerase?
    2. What is the fidelity of Q5® High-Fidelity DNA Polymerase?
    3. How should I determine an appropriate annealing temperature for my reaction?
    4. What should my primer concentration be when using Q5® High-Fidelity DNA Polymerase products?
    5. How should I set up a PCR experiment using Q5® High-Fidelity DNA Polymerase?
    6. My template is GC rich or supercoiled. How can I optimize my product yield using Q5® High-Fidelity DNA Polymerase?
    7. Do I need to modify my annealing temperature when using the Q5® High GC Enhancer?
    8. When should I add the High GC Enhancer?
    9. Are the DNA fragments produced by Q5® High-Fidelity DNA Polymerase blunt-ended or do they have the single-base 3´ overhang that Taq DNA Polymerase yields?
    10. There is a precipitate in the bottom of the buffer tube. Is this normal?
    11. What length of product can be made by Q5® High-Fidelity DNA Polymerase?
    12. I am having trouble amplifying a template that is longer than 5kb. How can I optimize my product yield using Q5® High-Fidelity DNA Polymerase?
    13. Does Q5® High-Fidelity DNA Polymerase exhibit a strand displacement activity?
    14. Where can I find help troubleshooting my PCR?
    15. Will Q5® High-Fidelity DNA Polymerase incorporate dUTPs?
    16. I'd like to clone a fragment amplified with Q5® High-Fidelity DNA Polymerase. Do I have to blunt-end clone?
    17. Do other polymerases work in Q5® Reaction Buffer?
    18. I am competing in the iGEM competition. Do you have any products that I should consider purchasing from NEB?
    1. PCR Using Q5® High-Fidelity DNA Polymerase (M0491)

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